This invention relates to DNA sequencing and in particular to automated methods for DNA sequencing.
DNA sequencing is generally carried out by the method of Sanger et al. (Proc. Nat. Acad. Sci. USA 74:5463, 1977) and involves enzymatic synthesis of single strands of DNA from a single stranded DNA template and a primer. Referring to FIG. 1, four separate syntheses are carried out. A single stranded template is provided along with a primer which hybridizes to the template. The primer is elongated using a DNA polymerase, and each reaction terminated at a specific base (guanine, G, adenine, A, thymine, T, or cytosine, C) via the incorporation of an appropriate chain Terminating agent, for example, a dideoxynucleotide. Enzymes currently used for this method of sequencing include: the large fragment of Escherichia coli DNA polymerase I ("Klenow" fragment), reverse transcriptase, Taq polymerase, and a modified form of bacteriophage T7 DNA polymerase.
Still referring to FIG. 1, the four DNA synthesis reactions result in formation of four series of DNA products, each product having one defined terminus and one variable terminus. The defined terminus starts with the primer molecule. The variable terminus ends with a chain terminating agent specific for the nucleotide base (either G, A, T, or C) at which the synthesis reaction terminated. The four different series of products are each separated on the basis of their molecular weight, in four separate lanes in a high resolution polyacrylamide gel, to form four series of bands, with each band on the gel corresponding sequentially to a specific nucleotide in the DNA sequence. Thus, the relative positions of the bands identify the positions in the DNA sequence of each given nucleotide base. Generally, the DNA products are labelled so that the bands produced are readily detected. As shown in FIG. 1, the intensity of the bands is generally non-uniform, within a single lane, because band intensity is directly related to the total number or concentration of DNA products of the same molecular weight in a specific lane, and this number varies from one product to another even when they are of approximately the same molecular weight and even when they contain the same chain terminating agent.
Using the above methodology, automated systems for DNA sequence analysis have been developed. One instrument, manufactured by EG&G, uses a .sup.32 P-label and a DNA polymerase, and the resulting DNA products separated by gel electrophoresis. Toneguzzo et al., 6 Biotechniques 460, 1988. A .sup.32 P-detector at the bottom of the gel scans for radioactivity as it passes through the bottom of the gel. Four synthesis reactions are required for each template to be sequenced, as well as four lanes on each gel, a separate lane being used for products terminated by each specific chain terminating agent, as shown for example in FIG. 1.
Kanbara et al., 6 Biotechnology 816, 1988, have replaced the .sup.32 P-labelled primer, described above, with a fluorescent-labelled primer. The resulting fluorescently labelled products are excited with a laser at the bottom of the gel and the fluorescence detected with a CRT monitor. This procedure also requires four 90% synthesis reactions and four lanes on the gel for each template to be sequenced.
Applied Biosystems manufactures an instrument in which four different primers are used, each labelled with a different fluorescent marker. Smith et al., 13 Nuc. Acid. Res. 2399, 1985; and 321 Nature 674, 1986. Each primer is used in a separate reaction containing one of four dideoxynucleotides. After the four reactions have been carried out they are combined together and run in a single lane on a gel. A laser at the bottom of the gel is used to detect fluorescent products after they have been permeated or electrophoresed through the gel. This system requires four separate annealing reactions and four separate synthesis reactions for each template, but only a single lane on the gel. Computer analysis of the sequence is made easier by having all four bands in a single lane.
DuPont provides an instrument in which a different fluorescent marker is attached to each of four dideoxynucleoside triphosphates. Prober et al., 238 Science 336, 1987. A single annealing step, a single polymerase reaction (containing each of the four labelled dideoxynucleosides triphosphates) and a single lane in the sequencing gel are required. The four different fluorescent markers in the DNA produces are detected separately as they are electrophoresed through the gel.
Englert et al., U.S. Pat. No. 4,707,237 (1987), describes a multichannel etectrophoresis apparatus having a detection means, disposed substantially across the whole width of the gel, which can sense labelled DNA products as they migrate past the detector means in four separate lanes, and identifies the channel or lane in which the sample is located. Preferably, radioisotopic labels are used.
Inherent to procedures currently used for DNA sequence analysis is the necessity to separate either radioactively or fluorescently-labelled DNA products by a gel permeation procedure such as polyacrylamide or to other gel electrophoresis, and then detect their locations relative to one another along the axis of permeation or movement through the gel. The accuracy of this procedure is determined in part by the uniformity of the signal in bands which have permeated approximately the same distance through the gel. Differences or variations in signal intensities between nearby bands create several problems. First, they decrease the sensitivity of the method, which is limited by the ability to detect the bands containing the weakest signals. Second, they create difficulties in determining whether a band with a weak signal is a True signal due to the incorporation of a chain terminating agent, or an artifact due to a pause site in the DNA, where the polymerase has dissociated. Third, they decrease the accuracy in determining the DNA sequence between closely spaced bands since the strong signal of one band may mask the weak signal of its neighbor.